Recordings from dissociated LNvs expressing GCaMP1.6 were carried out as in Dahdal et al. (2010). Briefly, 30–60 larval brains were dissociated by treatment with 2 units/ml Dispase II and manual trituration. GCaMP fluorescence from individual neurons was imaged on an inverted epifluorescence microscope (TE2000U, Nikon) via a standard GFP filter set. Cells were continuously superfused at 2 ml/min

with standard saline (128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 1.8 mM CaCl2, 36 mM sucrose, and 5 mM HEPES [pH 7.1]), to which compounds were added as indicated. For low-chloride experiments, standard saline was modified to reduce Cl− Gemcitabine order to 13.6 mM by replacement of NaCl with sodium gluconate. For locomotor activity experiments, adults were entrained to 12:12 LD cycles at 25°C for at least 3 days before transfer to DD. Locomotor activity was recorded

by using the DAM system (TriKinetics). We used χ2 analysis in ClockLab (Actimetrics) to derive a power and significance for each rhythm over 10 days in DD. We subtracted the significance score from the power to calculate the strength of each rhythm (presented as “power” in Results). Using this analysis, we found that control lines have average powers ranging from ∼270–580 (“rhythmic,” see Table 1), whereas classical clock mutants (per01, ClkJrk, and Clkar) have powers from 10–40 (“arrhythmic”). Pdf > dORKΔC flies, previously described as ∼70% arrhythmic / 30% find more weakly rhythmic ( Nitabach et al., 2002 and Wu et al., 2008), have an average power of 91, establishing a baseline for the effect of manipulations of electrical excitability. All statistical comparisons were many made by ANOVA. We are very grateful to the following for their generous gifts of antibodies and flies: Ravi Allada, Patrick Emery, Paul Hardin, Rob Jackson, Michael Nitabach, Jae Park, Marie-Laure Parmentier, Michael Rosbash, Amita Sehgal, and Mike Young. Additional fly stocks were obtained from the Vienna

Drosophila RNAi and Bloomington Stock centers, and PDF antisera were obtained from the DSHB. We also thank Afroditi Petsakou for advice on qPCR and Ravi Allada, Matthieu Cavey, Claude Desplan, Bambos Kyriacou, and Afroditi Petsakou for helpful comments on the manuscript. B.C. was supported by The Robert Leet and Clara Guthrie Patterson Trust Postdoctoral Fellowship, Bank of America, Trustee. E.A.K. was supported by a Dean’s Undergraduate Research Fellowship from NYU. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant Number C06 RR-15518-01 from the National Center for Research Resources, National Institutes of Health (NIH). Confocal microscopy was performed in the NYU Center for Genomics and Systems Biology Core Facility. This work was supported by NIH grants NS030808 (M.H.A.) and GM063911 (J.B.).